Several eukaryotic DNA viruses maintain their genomes as extrachromosomal multicopy nuclear episomes in proliferating host cells. Such episomal maintenance is characteristic of latent infection of the Bovine papillomavirus type 1 (BPV-1), Epstein-Barr Virus (EBV) as well as for Kaposi sarcoma associated Human herpesvirus type 8 (HHV8). The latency of the viral genome in dividing cell population requires activity of the viral genome at the two phases of the cell cycle: the viral genome replication during the S phase and proper segregation and partitioning of the replicated genomes into daughter cells during the host cell mitosis. For BPV-1 and two members of the gammaherpesvirus family—EBV and HHV8 an effective segregation of viral genomes into daughter cells and nuclear retention during mitosis is mediated through a single viral protein serving as a molecular linker, which attaches viral genomes to the host mitotic chromosomes. This linker protein is a viral regulatory protein E2 for BPV-1, viral transactivator EBNA1 for EBV and viral transcription repressor LANA1 for HHV8.
For initiation of the BPV-1 DNA replication in vivo, minimal origin region in cis and two viral proteins—E1 and E2, in trans, are absolutely essential. However, the minimal origin (MO) is not sufficient for stable extrachromosomal replication in dividing cells. An additional element, the Minichromosome Maintenance Element (MME) ensures the long-term episomal persistence of the genome in the presence of viral E1 and the E2 proteins in the dividing cells. In the BPV-1 genome, total of 17 E2 protein binding sites with different affinities to E2 can be identified: 12 of these are locating in the noncoding upstream regulatory region (URR). We have shown that the minichromosome maintenance element (MME) activity can be provided by the subset of the E2 binding sites. The function of multimeric E2 protein binding sites in the stable maintenance of the BPV-1 genomes is to provide the anchoring function for the E2 protein, which therefore tethers MME containing plasmids to mitotic chromosomes. This linkage between the BPV-1 genome and host chromatin ensures also that the viral genome is targeted to the nucleus when the nuclear membrane is reassembled during mitosis. In the case of EBV, the stable maintenance of replicated genomes is achieved due to the EBNA 1 protein and FR-element, which is comprised of multimeric EBNA1 protein binding sites.
We have shown that the BPV 1 E2 protein dependent MME (Abroi et al. 2004) and EBV EBNA1 dependent FR (Männik, Janikson and Ustav, unpublished) segregation/partitioning activities function independently from replication of the plasmids.
Transfection or infection of permissive cells with polyomavirus genome or replicator results in amplificational replication leading to cell death due to the over-replication. The mechanism of the BPV-1 origin based episomal replication is more complex and controlled. On one hand the first amplificational replication step, resembling in many aspects polyomavirus lytic over-replication is crucial for establishment of the stable episomal replication of the papillomavirus DNA. Such replication leads to increase of expression level of the viral proteins and copy-number of the viral genome. Increase of the E1 protein concentration, however, over certain limit induces the “onion-skin” type replication of the BPV-1 origin and generation of the replication intermediates having tendency for high frequency of DNA rearrangements and integration of the fragments of the viral DNA into chromosomal DNA. To maintain the stability and intactness of the viral genome, virus has to apply certain mechanism to assure proper balance between initiation and elongation of replication fork as well as segregation/partitioning of the viral plasmids during cell division
Therapeutic protein production in small and large scale is important field of development in pharmaceutical industry, because proteins produced in animal cells have proper processing, post-translational modification and therefore have adequate activity for treatment of the physiological condition. In general, for research purposes, the transient expression systems are used. The expression plasmids equipped with strong promoter driving expression of gene of interest is transfected into the appropriate cells using either chemical transfection to (like Lipofectamine 2000) or physical transfection, like electroporation. Transfection could be carried out in small scale, resulting in small amount of produced protein or transfected in large scale (up to 100 liters of cell suspension) allowing harvesting expressed protein in large quantity. Problem with large scale transfections is high cost for expensive transfection agent, large quantity of the expression plasmid, and high cost for maintaining large quantity of the cell culture. In these cases the transient transfection has been optimized for 293 HEK cells. However, therapeutic proteins for human use are mostly produced in Chinese Hamster Ovary (CHO cells), which have been proved to be safe and effective for production of therapeutic proteins. This is achieved by generating CHO stable super-producer cell lines isolated as result of screening and several subclonings. These steps are time and money consuming and therefore impractical for research applications. Use of CHO cells has turned out to be difficult in transient production assays due to the poor transfection and production capability.
In order to overcome the shortage of CHO cells in production of therapeutic proteins in transient transfections, use of episomal expression vectors is one of the possibilities enhancing expression plasmid copy-number and maintaining it in the cells for extended time for enlarging fraction of cell producing therapeutic protein as well as improving yield of the protein production.
The stable episomal maintenance systems described earlier (U.S. Pat. No. 6,479,279) were provided with homologous replication origins. Characteristic for these systems is for example a high mutation frequency, especially recombination. Furthermore, the system does not maintain stably replicating episomes in cells because part of the cells lose their plasmid in every generations. This deficiency of the system can be compensated by application of continuous antibiotic selection pressure on cells in order to eliminate the plasmid-negative cells from the culture. This fact creates serious limits for the system to be used for example in protein production.
U.S. patent application Ser. No. 10/938,864 (Kunaparaju) provides a heterologous system, which is capable of stable episomal replication lasting a couple of weeks. Kunaparaju uses two functional replicons in the expression plasmid—one dependent on wtPyV replication origin and Large T-antigen and second, oriP and EBNA1. Limitations in this system is use of wild type polyomavirus origin equipped with enhancer which we have shown to initiate over-replication and generating too high copy-number of the plasmid eventually leading to death of the cells. Cell adaptation to too high copy-number will include rearrangement of the plasmid leading to genetic instability and therefore incompatibility with the requirements for therapeutic protein production. Second limitation is use of complete oriP, which is comprised of two elements—Dyad Symmetry Element (DUE), which is eukaryotic origin of replication functioning as a result of cellular replication factors and Family of Repeats (FR), which is serving as cis-sequence for EBNA1 dependent segregation/partitioning of the plasmid. It means that Kunaparaju et al. invention uses two viral eukaryotic origins of replication, which may fire independently of each other and generate a conflict between initiation of DNA replication. The present disclosure provides improvements over the problems encountering prior systems.